U.S. patent application number 12/741514 was filed with the patent office on 2011-08-11 for streptococcus pyogenes classification.
This patent application is currently assigned to Novartis Vaccines and Diagnostics SRL. Invention is credited to Fabiana Falugi, Guido Grandi, Marirosa Mora, John L. Telford, Chiara Zingaretti.
Application Number | 20110195409 12/741514 |
Document ID | / |
Family ID | 38858212 |
Filed Date | 2011-08-11 |
United States Patent
Application |
20110195409 |
Kind Code |
A1 |
Grandi; Guido ; et
al. |
August 11, 2011 |
STREPTOCOCCUS PYOGENES CLASSIFICATION
Abstract
The difference Lancefield T-serotypes correlate with the
sequence of the pilus backbone protein (Pbp) in Streptococcus
pyogenes (GAS). We have sequenced Pbp for over 50 GAS strains,
representing the major disease-associated serotypes, and have
identified 15 Pbp variants. These 15 variants have been shown to
determine the specificity of the T-serotyping, such that sequencing
of the Pbp from a given GAS strain reliably predicts that strain's
T-serotype. thus the invention permits the t-serotype of a GAS
strain to be determined based on genotype.
Inventors: |
Grandi; Guido; (Milano,
IT) ; Mora; Marirosa; (Siena, IT) ; Telford;
John L.; (Monteriggioni, IT) ; Zingaretti;
Chiara; (Ravenna, IT) ; Falugi; Fabiana;
(Monteriggioni, IT) |
Assignee: |
Novartis Vaccines and Diagnostics
SRL
Siena
IT
|
Family ID: |
38858212 |
Appl. No.: |
12/741514 |
Filed: |
November 6, 2008 |
PCT Filed: |
November 6, 2008 |
PCT NO: |
PCT/GB2008/003769 |
371 Date: |
March 23, 2011 |
Current U.S.
Class: |
435/6.11 ;
252/182.12; 530/324; 530/325; 530/326; 530/327; 530/328; 530/329;
530/350; 530/387.9; 536/23.7 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12Q 2600/16 20130101 |
Class at
Publication: |
435/6.11 ;
530/329; 530/328; 530/327; 530/326; 530/325; 530/324; 530/350;
536/23.7; 530/387.9; 252/182.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 7/06 20060101 C07K007/06; C07K 14/315 20060101
C07K014/315; C07H 21/00 20060101 C07H021/00; C07K 16/12 20060101
C07K016/12; C09K 3/00 20060101 C09K003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 6, 2007 |
GB |
0721757.6 |
Claims
1. A method for determining the T-classification of a Streptococcus
pyogenes bacterium, wherein the sequence of the bacterium's pbp
gene is determined in whole or in part.
2. A method for analysing a Streptococcus pyogenes bacterium,
comprising a step in which the sequence of the bacterium's pbp gene
is determined in whole or in part.
3. A method for determining the T-classification of a Streptococcus
pyogenes bacterium, wherein the sequence, in whole or in part, of
the bacterium's pbp gene is compared to one or more known pbp
sequence(s) that have been correlated with T serotypes.
4. A kit for analysing a Streptococcus pyogenes bacterium,
comprising primers for amplifying a nucleic acid sequence
comprising the whole of part of a pbp gene from a Streptococcus
pyogenes bacterium.
5. A method for determining if a Streptococcus pyogenes bacterium
has a particular T serotype, comprising a step in which the
sequence of the bacterium's pbp gene is compared to the sequence of
a pbp gene from a Streptococcus pyogenes strain that is known to
have the particular T serotype, with a sequence match indicating
that the bacterium has the particular T serotype and no sequence
match indicating that the bacterium does not have the particular T
serotype.
6. A method for determining if a Streptococcus pyogenes bacterium
has a particular T serotype, comprising a step in which the
bacterium's pbp gene is contacted with a nucleic acid that, under
the conditions of this step, gives a first signal when contacted
with one of SEQ ID NOs: 58 to 70 and a second signal when contacted
with each of the other SEQ ID NOs: 58 to 70, wherein the first and
second signals can be distinguished from each other.
7. (canceled)
8. The method of claim 6 wherein the T serotype is T serotype
2.
9. The method of claim 6 wherein the T serotype is T serotype
3/13.
10. The method of claim 6 wherein the T serotype is T serotype
4.
11. The method of claim 6 wherein the T serotype is T serotype
5/27/44.
12. The method of claim 6 wherein the T serotype is T serotype
6.
13. The method of claim 6 wherein the T serotype is T serotype 8 or
25.
14. The method of claim 6 wherein the T serotype is T serotype
9.
15. The method of claim 6 wherein the T serotype is T serotype
11.
16. The method of claim 6 wherein the T serotype is T serotype
12.
17. The method of claim 6 wherein the T serotype is T serotype
14.
18. The method of claim 6 wherein the T serotype is T serotype
23.
19. The method of claim 6 wherein the T serotype is T serotype
3/5/13/28.
20. A kit comprising primers for amplifying a template sequence
comprising at least a part of the S. pyogenes pbp gene, the kit
comprising a first primer and a second primer, wherein the first
primer comprises a sequence substantially complementary to a
portion of said template sequence and the second primer comprises a
sequence substantially complementary to a portion of the complement
of said template sequence, wherein the sequences within said
primers which have substantial complementarity define the termini
of the template sequence to be amplified.
21. An isolated polypeptide selected from the group consisting of:
a polypeptide comprising an amino acid sequence having at least 50%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56 and 57; and a polypeptide comprising a
fragment of at least 7 consecutive amino acids of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4,
6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 and
57.
22. (canceled)
23. An isolated nucleic acid comprising a nucleotide sequence
encoding the polypeptides of claim 21.
24. An isolated antibody that (i) binds to only one of SEQ ID NOs:
1 to 13, and (ii) does not bind to Pap 1.
25. A mixture of a plurality of different isolated polypeptides
according to claim 21.
26. The method of claim 7 wherein the T serotype is T serotype 1.
Description
[0001] This application claims the benefit of United Kingdom patent
application 0721757.3 filed on 6 Nov. 2007, the complete contents
of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention is in the field of classifying Streptococcus
pyogenes (GAS) strains.
BACKGROUND ART
[0003] More than 50 years ago Lancefield and colleagues classified
GAS strains on the basis of serological recognition of the
trypsin-sensitive M antigen and of a trypsin-resistant antigen
known as the T antigen [1,2]. While the M-protein has been
thoroughly studied over the last three decades, the basis of
T-serotyping has not received the same attention.
[0004] There are about 20 known Lancefield T-serotypes (including
1, 2, 3, 4, 5, 6, 8, 9, 11, 12, 13, 14, 18, 22, 23, 25, 27, 28, 44,
B.sub.3264 and Impetigo 19), some of which are overlapping or
redundant. The T antigen has been used, in conjunction with the M
antigen, to provide an additional tool for the sub-classification
of GAS strains by an agglutination assay in which T-specific sera
are used [3,4]. These T-typing sera are obtained after the
streptococci are treated with trypsin, which digests the
trypsin-sensitive protein molecules on the cell surface including
the M protein, leaving the T antigen exposed. Furthermore, the T
antigens form the basis of a major serological typing scheme that
is used for those streptococci producing either no or a
non-typeable M protein.
[0005] One problem with the T-serotyping system is that it relies
on (i) the ability to maintain viable GAS organisms, in order to
provide sufficient protein for analysis and (ii) good-quality,
well-characterized antisera [5]. Moreover, some strains are often
recognized by patterns of closely associated T sera rather than by
single serum (e.g. T3/13/B3264, T5/27/44, T8/25/Imp19), and other
strains may react non-specifically with many T sera leading to
agglutination and, depending on the intensity of trypsinization,
they may lose true T-protein reaction [6].
[0006] Reference 7 concludes that the Pbp, Pap1 and PrtF2 proteins
all contribute to the T-type of GAS.
DISCLOSURE OF THE INVENTION
[0007] The inventors have found that the different Lancefield
T-serotypes correlate with the sequence of the pilus backbone
protein (Pbp) in GAS. They have sequenced Pbp for over 50 GAS
strains, representing the major disease associated serotypes, and
have identified fifteen Pbp variants. Thirteen of these variants
have been shown to determine the specificity of the T-serotyping,
such that sequencing of the Pbp from a given GAS strain can predict
that strain's T-serotype. Thus the invention permits the
T-classification of a GAS strain to be determined based on
genotype. Gene sequence analysis of the Pbp gene is much simpler
than the existing serological assays.
[0008] The invention provides a method for determining the
T-classification of a Streptococcus pyogenes bacterium, wherein the
sequence of the bacterium's pbp gene is determined in whole or in
part. The invention also provides a method for analysing a
Streptococcus pyogenes bacterium, comprising a step in which the
sequence of the bacterium's pbp gene is determined in whole or in
part.
[0009] The invention also provides a method for determining the
T-classification of a Streptococcus pyogenes bacterium, wherein the
sequence, in whole or in part, of the bacterium's pbp gene is
compared to one or more known pbp sequence(s). Usually it is
compared to at least two known pbp sequences (e.g. at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more; preferably at least 5 sequences). If these known pbp
sequences have been correlated with T-serotypes then the closest
match between the bacterium's pbp sequence and the known pbp
sequence permits the bacterium's T-serotype to be determined.
[0010] The invention also provides a kit for analysing a
Streptococcus pyogenes bacterium, comprising primers for amplifying
a nucleic acid sequence comprising the whole of part of a pbp gene
from a Streptococcus pyogenes bacterium.
[0011] The sequence of the pbp gene can be compared to known
sequences and the T-classification can thereby be determined. As
described in more detail below, enough of the gene must be
sequenced to permit it to be distinguished from the pbp genes of
other T-serotypes.
[0012] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has a particular T-serotype,
comprising a step in which the sequence of the bacterium's pbp gene
is compared to the sequence of a pbp gene from a Streptococcus
pyogenes strain that is known to have the particular T-serotype,
with a sequence match indicating that the bacterium has the
particular T-serotype and no sequence match indicating that the
bacterium does not have the particular T-serotype. Further details
are provided below.
[0013] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has a particular T-serotype,
comprising a step in which the bacterium's pbp gene is contacted
with a nucleic acid that, under the conditions of this step, gives
a first signal when contacted with one of SEQ ID NOs: 58 to 70 and
a second signal when contacted with each of the other SEQ ID NOs:
58 to 70, wherein the first and second signals can be distinguished
from each other. Thus, for example, the step may use a primer or
probe that is specific to only one of SEQ ID NOs: 58 to 70, with
amplification or hybridisation being the first signal and lack of
amplification or hybridisation being the second signal. This method
may be performed directly on S. pyogenes nucleic acid, but will
usually be performed on nucleic acid amplified therefrom. The
second signal may include a variety of different sub-signals, but
each of these can be distinguished from the first signal.
[0014] A relationship between sequence and T-serotype has
previously been suggested. Schneewind et al. [8] cloned a gene
coding for a protein recognized by the T6 antisera and located the
gene in the FCT (Fibronectin-binding, Collagen-binding T-antigen)
region of a M6 strain. Reference 9 reports that the T6 antigen is
Pbp and that three variants of this protein are specifically
recognized by three other T antisera. However, the extent of Pbp
variability remained unclear until the present work, and nor was
the relationship of Pbp variation to Lancefield T-serotypes fully
understood. Even so, in some embodiments, the invention does not
relate to the analysis of a strain with one of the six following
T-serotypes: 1; 5; 6; 12; 27; 44. T-serotypes 5, 27 and 44 are
closely related.
GAS Tee-Types
[0015] The inventors have sequenced the pbp gene from over 50 GAS
strains and have identified 15 distinct variants. The prototype
amino acid sequences for each of the variants are SEQ ID NOs 1 to
15, encoded by SEQ ID NOs 58 to 72, respectively. 13 of the 15
variants have been correlated with T-serotypes as follows:
TABLE-US-00001 SEQ ID 1 2 3 4 5 6 7 8 9 10 11 12 13 T-type 1 2
3.sup.a 4 5.sup.b 6 8.sup.c 9 11 12 14 23 28 .sup.aThis variant
also seen in T-serotype 13 .sup.bThis variant also seen in
T-serotypes 27 and 44 .sup.cThis variant also seen in T-serotype
25
[0016] Sequence identity of at least 90% at the amino acid level
has been observed within each of these 13 variants, but between the
different variants it is generally less than 72% (see Table
II).
[0017] Because of the intra-variant conservation and inter-variant
variation, the sequence of a bacterium's pbp gene can readily be
placed into one of these 13 groups, and thereby its
T-classification can be assessed. In the event that a particular
strain does not fit into any of the 13 groups then, in a similar
way to the current T-system, it can be classified, at least
preliminarily, as being either in one of the T-types not shown
above or as being non-typeable.
[0018] A pbp gene may be sequenced in whole or in part. If partial
sequence is used then its size and location within the pbp gene
must be sufficient to place it in one (or none) of the 13 variants.
The alignments in FIG. 4 shows the regions of pbp that are
conserved between variants (i.e. unsuitable for distinguishing
between them) and those that vary (i.e. can be used to distinguish
the 13 variants). FIG. 4 aligns the variants within FCT types,
because sequences in different FCT types align poorly and thus can
readily be distinguished, whereas sequences in the same FCT type
show several regions of overlap. For examples the first 180 and
last 125 residues of the sequences encoding SEQ ID NOs: 3, 5, 8, 9,
10, 11, 13 and 14 are very conserved (FIG. 4A) and so a typical
method to distinguish these sequences will focus on different
regions. The coding sequences for SEQ ID NOs: 1 and 4 are
sufficiently different from each other and from the other SEQ ID
NOs: that they can readily be distinguished.
[0019] In some cases more than one partial sequence may be
determined, such that the combination of the two partial sequences
is enough to determine the sequence's variant type, even though
each individual partial sequence might, on its own, not be
enough.
[0020] Where the invention refers to comparing the sequence of two
nucleic acid sequences, this comparison may be performed at a
nucleic acid level, or may be performed after transforming the
sequence e.g. by comparing inferred amino acid sequences encoded by
the two nucleic acids, or by comparing complements and reverse
complements, etc.
Nucleic Acid Detection
[0021] Various methods are known in the art for the
sequence-specific detection of nucleic acids. Any of these can be
used with the invention.
[0022] One of the advantages of using nucleic acid for identifying
the T-serotype of a strain rather than immunological techniques is
that, because efficient nucleic acid amplification techniques are
widely available, viable GAS organisms do not have to be maintained
in order to provide sufficient material for analysis. On the
contrary, nucleic acids can be amplified and detected even with
very low amounts of original GAS material.
[0023] Thus a method of the invention may involve a step of
amplifying nucleic acid present in a sample. Suitable techniques
include PCR, SDA, SSSR, LCR, TMA, NASBA, T7 amplification, etc. The
technique preferably gives exponential amplification. The technique
may be quantitative and/or real-time. Kits and methods for
amplification and detection of bacterial sequences are known in the
art e.g. it is known to characterise GAS strains by emm-specific
PCR [10]. Array-based techniques can also be used.
[0024] Amplification techniques generally involve the use of at
least one primer. With two primers and a double-stranded target,
the primers hybridize to different strands of the target and are
then extended. The extended products then serve as targets for
further rounds of hybridization/extension, permitting exponential
amplification. The net effect is to produce an amplicon from the
target, the 5' and 3' termini of the amplicon being defined by the
locations of the two primers in the target.
[0025] Thus the invention provides a kit comprising primers for
amplifying a template sequence comprising at least a part of the S.
pyogenes pbp gene, the kit comprising a first primer and a second
primer, wherein the first primer comprises a sequence substantially
complementary to a portion of said template sequence and the second
primer comprises a sequence substantially complementary to a
portion of the complement of said template sequence, wherein the
sequences within said primers which have substantial
complementarity define the termini of the template sequence to be
amplified.
[0026] Kits and methods may use primers that are specific to one
pbp variant, meaning that amplification will occur when that
variant is present in a sample but will not occur if the variant is
absent (e.g. if a different pbp variant is present). In other
embodiments, they may use primers that are not specific to any
particular pbp variant, meaning that amplification will occur when
various pbp variants are present in a sample. Where such
non-variant-specific primers are used then the variants can be
distinguished from each other by characterising the amplicons e.g.
by means of variant-specific probes, by sequencing the amplicons,
etc. Examples of variant-specific primers are given below.
[0027] Primers for amplifying sequences from the pbp gene may be
located inside the gene or outside the gene, provided that their
amplicon comprises the whole or part of the pbp gene. FIG. 1 shows
the genetic environment of the pbp gene in different FCT types, and
primers outside the pbp gene may be located accordingly.
[0028] Kits of the invention may further comprise primers and/or
probes for generating and detecting an internal standard, in order
to aid quantitative measurements.
[0029] Kits of the invention may further comprise a probe which is
substantially complementary to the template sequence and/or to its
complement and which can hybridize thereto. This probe can be used
in a hybridization technique to detect an amplicon. Such a probe
may be variant-specific.
[0030] Kits of the invention may comprise more than one pair of
primers (multiplex). Multiple pairs can be used for nested
amplification of a target sequence, or can be used to amplify
different target sequences. For instance, a kit or method may use a
plurality of primer pairs, each pair permitting amplification of
different pbp variants, thereby ensuring that a single set of
reagents can amplify a range of different pbp variants. Where a
plurality of primer pairs is used, it is possible to have a common
primer in two pairs, but at least one primer will differ in each
pair, thereby giving different amplicons.
[0031] Kits of the invention may also include one or more reagents
for determining a strain's M-type. Kits and reagents for emm-typing
are commercially available.
[0032] Because of the nature of nucleic acid hybridisation, if a
primer(s) or probe is used that is specific to a particular pbp
gene, a positive signal (e.g. generation of an amplicon, or
hybridisation to a probe) means that the sequence of that
particular gene has been determined in whole or in part, without
actual base-by-base sequencing. Such sequence-specific reagents
thus provide indirect sequence determination as a result of the
sequence-specific nature of their behaviour.
[0033] Example primers include SEQ ID NO:s 115-160, as shown in
Table I.
Tee-Type Detection
[0034] The invention provides a method for determining if a test
GAS has a particular T-serotype by comparing the sequence of its
pbp gene to the sequence of a pbp gene from a GAS that has a known
T-serotype. If the sequence from the test GAS matches the known
sequence then they have the same T-serotype; if the sequences do
not match then they have different T-serotypes.
[0035] The invention thus provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 1, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 1 (e.g. to SEQ ID NO: 58), with a match indicating that
the bacterium has T-serotype 1 and no match indicating that the
bacterium does not have T-serotype 1.
[0036] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 2, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 2 (e.g. to SEQ ID NO: 59), with a match indicating that
the bacterium has T-serotype 2 and no match indicating that the
bacterium does not have T-serotype 2.
[0037] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 3 or 13, comprising
a step in which the sequence of the bacterium's pbp gene is
compared to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 3 or 13 (e.g. to SEQ ID NO: 60), with a match indicating
that the bacterium has T-serotype 3 or 13 and no match indicating
that the bacterium does not have T-serotype 3 or 13.
[0038] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 4, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 4 (e.g. to SEQ ID NO: 61), with a match indicating that
the bacterium has T-serotype 4 and no match indicating that the
bacterium does not have T-serotype 4.
[0039] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 5, 27 or 44,
comprising a step in which the sequence of the bacterium's pbp gene
is compared to the sequence of a pbp gene from a S. pyogenes strain
in T-serotype 5, 27 or 44 (e.g. to SEQ ID NO: 62), with a match
indicating that the bacterium has T-serotype 5, 27 or 44 and no
match indicating that the bacterium does not have T-serotype 5, 27
or 44.
[0040] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 6, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 6 (e.g. to SEQ ID NO: 63), with a match indicating that
the bacterium has T-serotype 6 and no match indicating that the
bacterium does not have T-serotype 6.
[0041] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 8 or 25, comprising
a step in which the sequence of the bacterium's pbp gene is
compared to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 8 or 25 (e.g. to SEQ ID NO: 64), with a match indicating
that the bacterium has T-serotype 8 or 25 and no match indicating
that the bacterium does not have T-serotype 8 or 25.
[0042] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 9, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 9 (e.g. to SEQ ID NO: 65), with a match indicating that
the bacterium has T-serotype 9 and no match indicating that the
bacterium does not have T-serotype 9.
[0043] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 11, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 11 (e.g. to SEQ ID NO: 66), with a match indicating that
the bacterium has T-serotype 11 and no match indicating that the
bacterium does not have T-serotype 11.
[0044] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 12, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 12 (e.g. to SEQ ID NO: 67), with a match indicating that
the bacterium has T-serotype 12 and no match indicating that the
bacterium does not have T-serotype 12.
[0045] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 14, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 14 (e.g. to SEQ ID NO: 68), with a match indicating that
the bacterium has T-serotype 14 and no match indicating that the
bacterium does not have T-serotype 14.
[0046] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 23, comprising a
step in which the sequence of the bacterium's pbp gene is compared
to the sequence of a pbp gene from a S. pyogenes strain in
T-serotype 23 (e.g. to SEQ ID NO: 69), with a match indicating that
the bacterium has T-serotype 23 and no match indicating that the
bacterium does not have T-serotype 23.
[0047] The invention also provides a method for determining if a
Streptococcus pyogenes bacterium has T-serotype 3, 5, 13 or 28,
comprising a step in which the sequence of the bacterium's pbp gene
is compared to the sequence of a pbp gene from a S. pyogenes strain
in T-serotype 3, 5, 13 or 28 (e.g. to SEQ ID NO: 70), with a match
indicating that the bacterium has T-serotype 3, 5, 13 or 28 and no
match indicating that the bacterium does not have T-serotype 3, 5,
13 or 28.
[0048] Where the test GAS's pbp sequence is compared to the known
pbp sequence, this comparison may be against one of the known pbp
sequences disclosed herein (e.g. against a sequence encoding one of
SEQ ID NOs: 1 to 13), or it may be against a different known pbp
sequence that has sequence identity to one of the SEQ ID NOs: 1 to
13 coding sequences. Because of the low level of inter-variant
sequence identity, the comparison sequence can differ substantially
from SEQ ID NOs: 1 to 13 while still providing a useful result. For
instance, SEQ ID NO: 1 has .ltoreq.40% identity to the other 12
sequenced pbp variants and so the comparison sequence for
T-serotype I can code for SEQ ID NO: 1 or for a sequence having at
least 70% identity to SEQ ID NO: 1 (e.g. at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or more). The following table shows
this information for full-length SEQ ID NO: 1 and for the other SEQ
ID NOs 2 to 13:
TABLE-US-00002 SEQ ID Highest match Threshold e.g. at least % 1 40%
70% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 2 40% 70% 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% 3 72% 86% 88%, 90%, 95%, 96%,
97%, 98%, 99% 4 26% 63% 65%, 70% 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% 5 69% 85% 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% 6
55% 88% 90%, 95%, 96%, 97%, 98%, 99% 7 40% 70% 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% 8 60% 80% 85%, 90%, 95%, 96%, 97%, 98%, 99%
9 57% 79% 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 10 65% 83% 85%,
90%, 95%, 96%, 97%, 98%, 99% 11 67% 84% 85%, 90%, 95%, 96%, 97%,
98%, 99% 12 55% 88% 90%, 95%, 96%, 97%, 98%, 99% 13 72% 86% 88%,
90%, 95%, 96%, 97%, 98%, 99%
[0049] This table also shows how a sequence match may be defined.
If a comparison is being made against a pbp gene known to have
T-type 1 (e.g. SEQ ID NO: 1, encoded by SEQ ID NO: 58) then a
sequence identity of at least 70% can be considered as a match. In
contrast, If a comparison is being made against a pbp gene known to
have T-type 28 (e.g. SEQ ID NO: 13, encoded by SEQ ID NO: 70) then
a sequence identity of at least 70% would not be a match.
[0050] If comparison is being made indirectly, by determining if a
hybridisation event takes place (rather than by direct comparison
of sequence information), these figures for inter-variant sequence
identity can be used as the basis of stringency conditions for the
hybridisation. For instance, SEQ ID NO: 58 (T-type 1) has no more
than 40% identity to the pbp gene from any other T-type at an amino
acid level, and so stringency conditions can be selected that will
permit a primer or probe to hybridise to a target even when there
are substantial differences. In contrast, higher stringency should
be used with SEQ ID NO: 70 so as to avoid hybridisation with the
pbp sequences from other T-types.
Type-Specific Nucleic Acid Reagents
[0051] The invention provides a method for determining if test GAS
bacterium has a particular T-serotype in which its pbp gene
(including amplicons of the whole or part thereof) is contacted
with a type-specific nucleic acid reagent i.e. a reagent that gives
a particular signal when if encounters a nucleic acid target of a
desired pbp variant but gives a different signal (e.g. no signal)
when if encounters a nucleic acid target of a different pbp
variant.
[0052] For instance, if the reagent were contacted (under the same
hybridisation conditions) with each of SEQ ID NOs: 1 to 13 then it
would give a particular signal for one of these thirteen target
sequences but would give a different signal for the other thirteen.
Thus the presence of this signal will indicate that the relevant
target is present.
[0053] For example, the reagent may be a probe that can hybridise
to only one of SEQ ID NOs: 58 to 70. The reagent may be a primer
that can hybridise to only one of SEQ ID NOs: 58 to 70, or that has
a 3' sequence that permits extension only when it is hybridised to
a particular one of SEQ ID NOs: 58 to 70. Thus the reagent may be
used in combination with one or more further reagent(s) e.g. with a
second primer.
[0054] Examples of variant-specific primers are indicated in FIG.
4. These can be used in their indicated orientation or in reverse
complement orientation, as required.
[0055] FIG. 4A shows a forwards primer (SEQ ID NO: 161) that is
common to the coding sequences of SEQ ID NOs: 3, 5, 8, 9, 10, 11,
13 and 14. This is used in combination with seven different reverse
primers, as follows:
TABLE-US-00003 Target 3 5 8 9 10 11/14 13 Rev primer SEQ ID: 166
165 168 162 163 167 164 Amplicon length 685 530 865 145 308 767
395
[0056] Where the target sequence is in the same variant as SEQ ID
NO: 11 or 14 then the primers will amplify in the same way. A probe
specific to the region around nucleotides 700 of these two SEQ ID
NOs can then be used to distinguish them.
[0057] FIG. 4B shows a reverse primer (SEQ ID NO: 169) that is
common to the coding sequences of SEQ ID NOs: 2 and 7. This is used
in combination with two different forward primers, as follows:
TABLE-US-00004 Target 2 7 Fwd primer SEQ ID: 170 171 Amplicon
length 1674 1465
[0058] FIG. 4C shows a forwards primer (SEQ ID NO: 172) that is
common to the coding sequences of SEQ ID NOs: 6 and 12. This is
used in combination with two different reverse primers, as
follows:
TABLE-US-00005 Target 6 12 Rev primer SEQ ID: 173 174 Amplicon
length 1144 1245
[0059] Primer pair SEQ ID NOs 175 & 176 can be used to amplify
the coding sequence of SEQ ID NO: 5.
[0060] Primer pair SEQ ID NOs 177 & 178 can be used to amplify
the coding sequence of SEQ ID NO: 2.
[0061] The invention also provides a nucleic acid probe that can
hybridise to only one of SEQ ID NOs: 58 to 70. The invention also
provides a nucleic acid amplification primer that can hybridise to
only one of SEQ ID NOs: 58 to 70. The invention also provides a
nucleic acid amplification primer that has a 3' sequence that
permits extension when it is hybridised to only one of SEQ ID NOs:
58 to 70. The invention also provides a nucleic acid amplification
primer that has a 3' or 5' sequence that permits ligation when it
is hybridised to only one of SEQ ID NOs: 58 to 70. These
variant-specific probes and primers thus permit the 13 different
pbp variants to be uniquely identified.
Polypeptides
[0062] The invention provides a polypeptide comprising an amino
acid sequence having at least a % sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,
242, 243, 244, 245, 246, 247, 248, 249, 250, 251, and 252. For each
of these SEQ ID NOs, the value of a may be independently selected
from 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5 or even
100. Within this list of SEQ ID NOs, numbers 1-57 are Pbp
sequences, 179-216 are Pap1 sequences, and 217-252 are Pap2
sequences.
[0063] The invention also provides a polypeptide comprising a
fragment of at least b consecutive amino acids of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,
191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 251, and 252. For each of
these SEQ ID NOs, the value of b may be independently selected from
5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40,
45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more. The
fragment, may comprise at least one T-cell or, preferably, a B-cell
epitope of the sequence. T- and B-cell epitopes can be identified
empirically (e.g. using PEPSCAN [11,12] or similar methods), or
they can be predicted (e.g. using the Jameson-Wolf antigenic index
[13], matrix-based approaches [14], TEPITOPE [15], neural networks
[16], OptiMer & EpiMer [17,18], ADEPT [19], Tsites [20],
hydrophilicity [21], antigenic index [22] or the methods disclosed
in reference 23, etc.).
[0064] A polypeptide of the invention may meet both the sequence
identity criterion and the fragment length criterion e.g. the
invention also provides a polypeptide comprising an amino acid
sequence having at least a % sequence identity to a particular SEQ
ID NO: and comprising a fragment of at least b consecutive amino
acids from that SEQ ID NO.
[0065] These polypeptides include homologs, orthologs, allelic
variants and mutants. Typically, 50% identity or more between two
polypeptide sequences is considered to be an indication of
functional equivalence. Identity between polypeptides is preferably
determined by the Smith-Waterman homology search algorithm as
implemented in the MPSRCH program (Oxford Molecular), using an
affine gap search with parameters gap open penalty=12 and gap
extension penalty=1.
[0066] Polypeptides of the invention may, compared to SEQ ID NOs:
1-57 or 179-252, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, etc.) conservative amino acid replacements i.e. replacements
of one amino acid with another which has a related side chain.
Genetically-encoded amino acids are generally divided into four
families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e.
lysine, arginine, histidine; (3) non-polar i.e. alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar i.e. glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified jointly as
aromatic amino acids. In general, substitution of single amino
acids within these families does not have a major effect on the
biological activity. The polypeptides may have one or more (e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions
relative to a reference sequence. The polypeptides may also include
one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions
(e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to a reference
sequence.
[0067] Polypeptides of the invention can be prepared in many ways
e.g. by chemical synthesis (in whole or in part), by digesting
longer polypeptides using proteases, by translation from RNA, by
purification from cell culture (e.g. from recombinant expression),
from the organism itself (e.g. after bacterial culture, or direct
from patients), etc. A preferred method for production of peptides
<40 amino acids long involves in vitro chemical synthesis
[24,25]. Solid-phase peptide synthesis is particularly preferred,
such as methods based on tBoc or Fmoc [26] chemistry. Enzymatic
synthesis [27] may also be used in part or in full. As an
alternative to chemical synthesis, biological synthesis may be used
e.g. the polypeptides may be produced by translation. This may be
carried out in vitro or in vivo. Biological methods are in general
restricted to the production of polypeptides based on L-amino
acids, but manipulation of translation machinery (e.g. of aminoacyl
tRNA molecules) can be used to allow the introduction of D-amino
acids (or of other non natural amino acids, such as iodotyrosine or
methylphenylalanine, azidohomoalanine, etc.) [28]. Where D-amino
acids are included, however, it is preferred to use chemical
synthesis. Polypeptides of the invention may have covalent
modifications at the C-terminus and/or N-terminus.
[0068] Polypeptides of the invention can take various forms (e.g.
native, fusions, glycosylated, non-glycosylated, lipidated,
non-lipidated, phosphorylated, non-phosphorylated, myristoylated,
non-myristoylated, monomeric, multimeric, particulate, denatured,
etc.).
[0069] Polypeptides of the invention are preferably provided in
purified or substantially purified form i.e. substantially free
from other polypeptides (e.g. free from naturally-occurring
polypeptides), particularly from other GAS or host cell
polypeptides, and are generally at least about 50% pure (by
weight), and usually at least about 90% pure i.e. less than about
50%, and more preferably less than about 10% (e.g. 5% or less) of a
composition is made up of other expressed polypeptides.
Polypeptides of the invention are preferably GAS polypeptides.
[0070] Polypeptides of the invention may be attached to a solid
support. Polypeptides of the invention may comprise a detectable
label (e.g. a radioactive or fluorescent label, or a biotin
label).
[0071] The term "polypeptide" refers to amino acid polymers of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. Polypeptides can occur as
single chains or associated chains. Polypeptides of the invention
can be naturally or non-naturally glycosylated (i.e. the
polypeptide has a glycosylation pattern that differs from the
glycosylation pattern found in the corresponding naturally
occurring polypeptide).
[0072] Polypeptides of the invention may be at least 40 amino acids
long (e.g. at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500 or more).
Polypeptides of the invention may be shorter than 500 amino acids
(e.g. no longer than 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 220, 240, 260, 280, 300, 350, 400 or 450 amino
acids).
[0073] The invention provides polypeptides comprising a sequence
--X--Y-- of --Y--X--, wherein: --X-- is an amino acid sequence as
defined above and --Y-- is not a sequence as defined above i.e. the
invention provides fusion proteins. Where the N-terminus codon of a
polypeptide-coding sequence is not ATG then that codon will be
translated as the standard amino acid for that codon rather than as
a Met, which occurs when the codon is translated as a start
codon.
[0074] The invention provides a process for producing polypeptides
of the invention, comprising culturing a host cell of to the
invention under conditions which induce polypeptide expression.
[0075] The invention provides a process for producing a polypeptide
of the invention, wherein the polypeptide is synthesised in part or
in whole using chemical means.
[0076] The invention provides a composition comprising two or more
polypeptides of the invention.
Nucleic Acids
[0077] The invention also provides a nucleic acid comprising a
nucleotide sequence encoding the polypeptides of the invention e.g.
SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 253, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,
268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,
281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,
294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,
307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,
320, 321, 322, 323, 324, 325 and 326. The invention also provides
nucleic acid comprising nucleotide sequences having sequence
identity to such nucleotide sequences. Such nucleic acids include
those using alternative codons to encode the same amino acid.
[0078] The invention also provides nucleic acid which can hybridize
to these nucleic acids. Hybridization reactions can be performed
under conditions of different "stringency". Conditions that
increase stringency of a hybridization reaction of widely known and
published in the art. Examples of relevant conditions include (in
order of increasing stringency): incubation temperatures of
25.degree. C., 37.degree. C., 50.degree. C., 55.degree. C. and
68.degree. C.; buffer concentrations of 10.times.SSC, 6.times.SSC,
1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl and 15 mM
citrate buffer) and their equivalents using other buffer systems;
formamide concentrations of 0%, 25%, 50%, and 75%; incubation times
from 5 minutes to 24 hours; 1, 2, or more washing steps; wash
incubation times of 1, 2, or 15 minutes; and wash solutions of
6.times.SSC, 0.1.times.SSC, 0.1.times.SSC, or de-ionized water.
Hybridization techniques and their optimization are well known in
the art [e.g. see refs 29 & 30, etc.].
[0079] Nucleic acid comprising fragments of these sequences are
also provided. These should comprise at least n consecutive
nucleotides from the sequences and, depending on the particular
sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40,
50, 60, 70, 80, 90, 100, 150, 200 or more).
[0080] The invention provides nucleic acid of formula
5'-X--Y--Z-3', wherein: --X-- is a nucleotide sequence consisting
of x nucleotides; --Z-- is a nucleotide sequence consisting of z
nucleotides; --Y-- is a nucleotide sequence consisting of either
(a) a fragment of a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101; 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,
265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,
278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,
291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,
304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,
317, 318, 319, 320, 321, 322, 323, 324, 325 and 326 or (b) the
complement of (a); and said nucleic acid 5'-X--Y--Z-3' is neither
(i) a fragment of either a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,
264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,
277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,
290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,
303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,
316, 317, 318, 319, 320, 321, 322, 323, 324, 325 and 326 nor (ii)
the complement of (i). The --X-- and/or --Z-- moieties may comprise
a promoter sequence (or its complement).
[0081] The invention includes nucleic acid comprising sequences
complementary to these sequences (e.g. for antisense or probing, or
for use as primers).
[0082] Nucleic acid according to the invention can take various
forms (e.g. single-stranded, double-stranded, vectors, primers,
probes, labelled etc.). Nucleic acids of the invention may be
circular or branched, but will generally be linear. Unless
otherwise specified or required, any embodiment of the invention
that utilizes a nucleic acid may utilize both the double-stranded
form and each of two complementary single-stranded forms which make
up the double-stranded form. Primers and probes are generally
single-stranded, as are antisense nucleic acids.
[0083] Nucleic acids of the invention are preferably provided in
purified or substantially purified form i.e. substantially free
from other nucleic acids (e.g. free from naturally-occurring
nucleic acids), particularly from other GAS or host cell nucleic
acids, generally being at least about 50% pure (by weight), and
usually at least about 90% pure. Nucleic acids of the invention are
preferably GAS nucleic acids.
[0084] Nucleic acids of the invention may be prepared in many ways
e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA)
in whole or in part, by digesting longer nucleic acids using
nucleases (e.g. restriction enzymes), by joining shorter nucleic
acids or nucleotides (e.g. using ligases or polymerases), from
genomic or cDNA libraries, etc.
[0085] Nucleic acid of the invention may be attached to a solid
support (e.g. a bead, plate, filter, film, slide, microarray
support, resin, etc.). Nucleic acid of the invention may be
labelled e.g. with a radioactive or fluorescent label, or a biotin
label. This is particularly useful where the nucleic acid is to be
used in detection techniques e.g. where the nucleic acid is a
primer or as a probe.
[0086] The term "nucleic acid" includes in general means a
polymeric form of nucleotides of any length, which contain
deoxyribonucleotides, ribonucleotides, and/or their analogs. It
includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA
analogs, such as those containing modified backbones (e.g. peptide
nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus
the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA,
recombinant nucleic acids, branched nucleic acids, plasmids,
vectors, probes, primers, etc. Where nucleic acid of the invention
takes the form of RNA, it may or may not have a 5' cap.
[0087] Nucleic acids of the invention comprise sequences, but they
may also comprise non-GAS sequences (e.g. in nucleic acids of
formula 5'-X--Y--Z-3', as defined above). This is particularly
useful for primers, which may thus comprise a first sequence
complementary to a nucleic acid target and a second sequence which
is not complementary to the nucleic acid target. Any such
non-complementary sequences in the primer are preferably 5' to the
complementary sequences. Typical non-complementary sequences
comprise restriction sites or promoter sequences.
[0088] Nucleic acids of the invention may be part of a vector i.e.
part of a nucleic acid construct designed for
transduction/transfection of one or more cell types. Vectors may
be, for example, "cloning vectors" which are designed for
isolation, propagation and replication of inserted nucleotides,
"expression vectors" which are designed for expression of a
nucleotide sequence in a host cell, "viral vectors" which is
designed to result in the production of a recombinant virus or
virus-like particle, or "shuttle vectors", which comprise the
attributes of more than one type of vector. Preferred vectors are
plasmids. A "host cell" includes an individual cell or cell culture
which can be or has been a recipient of exogenous nucleic acid.
Host cells include progeny of a single host cell, and the progeny
may not necessarily be completely identical (in morphology or in
total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation and/or change. Host cells
include cells transfected or infected in vivo or in vitro with
nucleic acid of the invention.
[0089] Where a nucleic acid is DNA, it will be appreciated that "U"
in a RNA sequence will be replaced by "T" in the DNA. Similarly;
where a nucleic acid is RNA, it will be appreciated that "T" in a
DNA sequence will be replaced by "U" in the RNA.
[0090] The term "complement" or "complementary" when used in
relation to nucleic acids refers to Watson-Crick base pairing. Thus
the complement of C is G, the complement of G is C, the complement
of A is T (or U), and the complement of T (or U) is A. It is also
possible to use bases such as I (the purine inosine) e.g. to
complement pyrimidines (C or T).
[0091] Nucleic acids of the invention can be used, for example: to
produce polypeptides; as hybridization probes for the detection of
nucleic acid in biological samples; to generate additional copies
of the nucleic acids; to generate ribozymes or antisense
oligonucleotides; as single-stranded DNA primers or probes; or as
triple-strand forming oligonucleotides.
[0092] The invention provides a process for producing nucleic acid
of the invention, wherein the nucleic acid is synthesised in part
or in whole using chemical means.
[0093] The invention provides vectors comprising nucleotide
sequences of the invention (e.g. cloning or expression vectors) and
host cells transformed with such vectors.
[0094] The invention also provides a kit comprising primers (e.g.
PCR primers) for amplifying a template sequence contained within an
GAS nucleic acid sequence, the kit comprising a first primer and a
second primer, wherein the first primer is substantially
complementary to said template sequence and the second primer is
substantially complementary to a complement of said template
sequence, wherein the parts of said primers which have substantial
complementarity define the termini of the template sequence to be
amplified. The first primer and/or the second primer may include a
detectable label (e.g. a fluorescent label).
[0095] For certain embodiments of the invention, nucleic acids are
preferably at least 7 nucleotides in length (e.g. 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70,
75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
225, 250, 275, 300 nucleotides or longer).
[0096] For certain embodiments of the invention, nucleic acids are
preferably at most 500 nucleotides in length (e.g. 450, 400, 350,
300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 80, 75, 70, 65,
60, 55, 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 nucleotides or
shorter).
[0097] Primers and probes of the invention, and other nucleic acids
used for hybridization, are preferably between 10 and 30
nucleotides in length (e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
Antisera
[0098] Existing T-typing sera are raised against streptococci that
have been treated with trypsin. As shown below, and also in ref. 7,
these sera recognise both the Pbp and Pap1 proteins. Because we
have shown that T-typing is based on the Pbp protein then it is now
possible to provide T-typing sera that recognise Pbp but do not
recognise Pap1. Thus the invention provides an antibody that (i)
binds to only one of SEQ ID NOs: 1 to 13, and (ii) does not bind to
Pap1. The lack of binding to the 13 other Pbp sequences permits
T-typing by using this antibody. The lack of binding to Pap1
distinguishes the antibody from existing T-typing sera.
[0099] Antibodies of the invention may be polyclonal or monoclonal.
Monoclonal antibodies are particularly useful in identification and
purification of the individual polypeptides against which they are
directed. Monoclonal antibodies of the invention may also be
employed as reagents in immunoassays, radioimmunoassays (RIA) or
enzyme-linked immunosorbent assays (ELISA), etc. In these
applications, the antibodies can be labelled with an
analytically-detectable reagent such as a radioisotope, a
fluorescent molecule or an enzyme. The monoclonal antibodies
produced by the above method may also be used for the molecular
identification and characterization (epitope mapping) of
polypeptides of the invention.
[0100] Antibodies of the invention are preferably provided in
purified or substantially purified form. Typically, the antibody
will be present in a composition that is substantially free of
other polypeptides e.g. where less than 90% (by weight), usually
less than 60% and more usually less than 50% of the composition is
made up of other polypeptides.
[0101] The invention also provides a collection (e.g. in the form
of a kit) of a plurality of antibodies, wherein each of said
plurality (i) binds to only one of SEQ ID NOs: 1 to 13, (ii) does
not bind to Pap1. Preferably the plurality includes at least two
antibodies, each of which binds to a different one of SEQ ID NOs: 1
to 13. The collection may further comprise antibodies that do not
meet criteria (i) and (ii). The collection may be used for T-typing
of GAS e.g. by immunoblot, by FACS, etc.
Multi Pilus Vaccines
[0102] It has been shown that immunization with pilus components of
the three major streptococcal pathogens (GAS, GBS and S.
pneumoniae) can confer type-specific protection. A combination of
different Pbp proteins may represent a viable vaccine capable of
giving broad coverage against the most important strains involved
in disease. 12 Pbp variants account for at least 24 of the 27 most
prevalent M-types, and so a combination of these variants could
protect against 98% of the circulating strains.
[0103] Thus the invention provides a mixture of a plurality of
different polypeptides of the invention. A combination of at least
two different Pbp variants (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
or more) may be used as a vaccine against S. pyogenes disease.
General
[0104] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0105] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0106] The term "about" in relation to a numerical value x means,
for example, x+10%.
[0107] Unless specifically stated, a process comprising a step of
mixing two or more components does not require any specific order
of mixing. Thus components can be mixed in any order. Where there
are three components then two components can be combined with each
other, and then the combination may be combined with the third
component, etc.
[0108] Identity between polypeptide sequences is preferably
determined by the Smith-Waterman homology search algorithm as
implemented in the MPSRCH program (Oxford Molecular), using an
affine gap search with parameters gap open penalty=12 and gap
extension penalty=1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] FIG. 1 shows the genetic environment of the pbp gene in
different FCT-type strains of GAS.
[0110] FIG. 2 shows how T-sera recognised recombinant Pbp from
various strains in western blot.
[0111] FIG. 3 shows the results of a normal T-typing assay for
strains classified by their pbp variant type.
[0112] FIG. 4 shows alignments of Pbp genes. FIG. 4A aligns FCT-3
and FCT-4 strains. FIG. 4B aligns FCT-6 and FCT-9 strains. FIG. 4C
aligns FCT-1 strains.
[0113] FIG. 5 shows results of PCR using variant-specific
primers.
[0114] FIG. 6 shows results of multiplex PCR using a mixture of
variant-specific primers.
MODES FOR CARRYING OUT THE INVENTION
Sequence Variation in the Pilus Backbone Protein (Pbp)
[0115] Genes coding for the pilus structural components (pbp, pap1
and pap2) were investigated for 57 different GAS strains with a
variety of FCT genotypes and M-serotypes. The primers used for PCR
amplification of the pilus regions are shown in Table I. The Pbp
genes for the 57 strains are SEQ ID NOs: 58 to 114, encoding amino
acid sequences SEQ ID NOs: 1 to 57 respectively.
[0116] These 57 sequences were found to group into 15 variants.
Prototypic sequences for each variant are SEQ ID NOs: 1 to 15.
Table II herein shows the amino acid identity derived from pairwise
comparisons between these 15 variants. Within a single M-type the
Pbp protein varied by no more than 2/100 amino acids between
strains, although the DNA sequence revealed several silent single
nucleotide differences. In four cases the same variant was found in
different M-types, again with greater than 98% identity.
[0117] Pbp from a M18-type strain (SEQ ID NO: 11) had 90% identity
to Pbp from a M49-type strain (SEQ ID NO: 14). Rather than classify
these sequences as different variants, immunological
cross-reactivity between the strains led to their being classified
as a single variant.
[0118] In contrast to the high level of intra-variant amino acid
sequence identity, between different Pbp variants there is much
less conservation, reaching a maximum of 72% in tested
sequences.
[0119] The sequences of the Pap1 protein (SEQ ID NOs 179-216,
encoded by SEQ ID NOs 253-290) did not divide as clearly into
distinct variants and a wider spread of pairwise identities were
observed.
[0120] The sequences of Pap2 protein (SEQ ID NOs 217-252, encoded
by SEQ ID NOs 291-326) did not correlate with T-types.
Different Backbone Proteins are Associated with Different
T-Serotypes.
[0121] From the analysis of M-types associated with each Pbp
variant, we hypothesized that M-types which share the same backbone
might also share the same T-serotype, independent of the FCT type
carried and ancillary protein sequences.
[0122] 14 pbp genes (SEQ ID NOs: 1 to 14) were cloned and expressed
in E. coli and the resulting purified recombinant proteins were
tested in immunoblot with each of the 21 commercially available
T-typing antisera. The results are summarised in FIG. 2. In most
cases each Pbp was recognized by a single T-typing serum, except
that the Pbp that reacted with serum T-28 also reacted
non-specifically with some other T antisera. The Pbp sequences from
M-49 and M-18 types, which have an amino acid identity of 90%, were
recognized by the same T serum hence justifying considering these
two proteins as a single Pbp variant. In three cases, a single Pbp
reacted with more than one T-serum. However, in each case the
positive sera belonged to the known families of related T-sera
which are usually grouped together (T3/13/B3264, T5/27/44,
T8/25/Imp19).
[0123] Thus there is a clear correlation between the T-serotype and
specific Pbp variants sharing homology of at least 90%.
[0124] T-antisera also recognised the Pap1 ancillary protein 1, but
did not recognise Pap2. Unlike Pbp, though, there was no close
correlation between T-type and Pap1 variants.
T-Serotype Agglutination Specificity Correlates with Pbp
[0125] Because of the correlation between T-serotype and Pbp
variants in the western blot experiments, the standard
agglutination reaction (on which T-typing is based) was performed
using various GAS strains which had already been classified
according to their Pbp variant. As shown in FIG. 3, a strict
correspondence between a Pbp variant and T-agglutination was
observed. Thus the sequence of the pbp gene is sufficient to
predict the T-type.
[0126] To test this idea, we looked at a strain (M50.sub.--4538)
that had not been tested in the western blot experiments. Its pbp
gene was sequenced and its T-type was predicted. Agglutination
confirmed that the prediction was correct.
[0127] T-typing sera 5, 27 and 44 are known to cross-react. These
three sera reacted with three strains that, while having different
M-types and different pap1 sequences, shared an identical pbp
sequence. So far we did not find a pbp protein that reacts with
T-typing sera 18 or 22, but the latter occurs in only 1/4000
strains [31].
[0128] Thus, although T-typing sera recognise both the pap1 and the
pbp genes, it is the pbp product that determines the
T-serotype.
PCR Detection of Tee-Types
[0129] Bacterial DNA from the following strains was incubated with
the relevant PCR primer pairs described above and selected from SEQ
ID NOs: 161 to 178. Results of amplification are shown in FIG. 5.
Lanes 1 and 20 include molecular weight markers and lane 18 is
empty. The other lanes show amplification from strains: (2) 2727;
(3) 3789; (4) 20010070; (5) 2728; (6) 20023465; (7) 4538; (8) 6180;
(9) 3776/27/44; (10) 5481/27/44; (11) 2724/13; (12) 8232; (13)
2720; (14) SF370; (15) 3040; (16) DS71; (17) 20010012/25; (19)
20010040. Amplification was successful with these primers for these
strains.
[0130] A multiplex experiment was also performed, in which a
mixture of all primers was used. The results are shown in FIG. 6.
Each lane shows results obtained with a single strain. The lanes
are ordered by amplicon sizes, which are as follows: 145
bp=M11(2727), M78(3789), M89(20010070); 308 bp=M12(2728),
M22(20023465), M50(4538); 395 bp=M28(4436); 530 bp=M5(4883),
M44(3776), M44(5481), M77(4959); 685 bp=M3(3040); 767 bp=M18(8232);
865 bp=M9(2720); 1024 bp=M1(SF370); 1144 bp=M6(2724); 1245
bp=M23(DSM2071); 1338 bp=M75(20010012); 1630 bp=M2(20010064); 2151
bp=M4(20010040). FIG. 6 confirms that the primers can amplify their
target strains even in the presence of primers for other
targets.
[0131] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
REFERENCES
[0132] [1] Lancefield, R. C., The antigenic complex of
Streptococcus hemolyticus. I. Demonstration of a type-specific
substance in extracts of Streptococcus hemolyticus. J Exp Med,
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properties of T antigen extracted from group A hemolytic
streptococci. J Exp Med, 1946. 84: p. 449-471. [0134] [3] Griffith,
F., The serological classification of streptococcus pyogenes. J.
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Tanner, J. Winship, R. Swans, K. M. Ries, and a.E.L.K. P. M.
Schlivert, Severe group A streptococcal infections associated with
a toxic shock-like syndrome and scarlet fever toxin A. N. Engl. J.
Med., 1989. 321: p. 1-7. [0136] [5] Neal, S., et al., International
Quality Assurance Study for Characterization of Streptococcus
pyogenes. J Clin Microbiol, 2007. 45(4): p. 1175-9. [0137] [6]
Johnson, D. R., et al., Characterization of group A streptococci
(Streptococcus pyogenes): correlation of M-protein and emm-gene
type with T-protein agglutination pattern and serum opacity factor.
J Med Microbiol, 2006. 55(Pt 2): p. 157-64. [0138] [7] Lizano et
al. (2007) J Bacteriol, 189(4):1426-34. [0139] [8] Schneewind, O.,
K. F. Jones, and V. A. Fischetti, Sequence and structural
characteristics of the trypsin-resistant T6 surface protein of
group A streptococci. J Bacteriol, 1990. 172(6): p. 3310-7. [0140]
[9] Mora, M., et al., Group A Streptococcus produce pilus-like
structures containing protective antigens and Lancefield T
antigens. Proc Natl Acad Sci USA, 2005. 102(43): p. 15641-6. [0141]
[10] Beall et al. (1996) J Clin Microbiol 34:953-8. [0142] [11]
Geysen et al. (1984) PNAS USA 81:3998-4002. [0143] [12] Carter
(1994) Methods Mol Biol 36:207-223. [0144] [13] Jameson, B A et al.
1988, CABIOS 4(1):181-186. [0145] [14] Raddrizzani & Hammer
(2000) Brief Bioinform 1(2):179-189. [0146] [15] De Lalla et al.,
(1999) J. Immunol. 163:1725-1729. [0147] [16] Brusic et al (1998)
Bioinformatics 14(2):121-130 [0148] [17] Meister et al. (1995)
Vaccine 13(6):581-591. [0149] [18] Roberts et al. (1996) AIDS Res
Hum Retroviruses 12(7):593-610. [0150] [19] Maksyutov &
Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-297. [0151] [20]
Feller & de la Cruz (1991) Nature 349(6311):720-721. [0152]
[21] Hopp (1993) Peptide Research 6:183-190. [0153] [22] Welling et
al. (1985) FEBS Lett. 188:215-218. [0154] [23] Davenport et al.
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Fields et al. (1997) Meth Enzymol 289: Solid-Phase Peptide
Synthesis. ISBN: 0121821900. [0157] [26] Chan & White (2000)
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TABLE-US-00006 [0162] TABLE I Strain Primer Primer Sequence SEQ ID
SF37C Pbp_for GTGCGTCATATGGCTACAACAGTTCACGG 115 Pbp_rev
GCGTCTCGAGAAAGTCTTTTTTATTTGTAAAAGTAATG 116 Pap1_for
GTGCGTCATATGGCTAAGACTGTTTTTGGT 117 Pap1_rev
GCGTCTCGAGGCCATTGATCTTTTGA 118 20010064 Pbp_for
GTGCGTCATATGGAGGACACCAGAGTGCCT 119 Pbp_rev
GCGTCTCGAGTGAAGGACGTTTGTTGTTTTTAACCGA 120 Pap1_for
GTGCGTGCTAGCGAAACTCAAGATAACAATCCAGCAC 121 Pap1_rev
GCGTCTCGAGAACACTCGGTGGACGTTTTG 122 3040 Pbp_for
GTGCGTCATATGGAGACGGCAGGAGTG 123 Pbp_rev
GCGTCTCGAGAGCTTTTTTACGTTTTGTAATATAG 124 Pap1_for
GTGCGTGCTAGCGCTGAAGAACAATCAGTG 125 Pap1_rev
GCGTCTCGAGAAGATCTTTTCGGTTTTC 126 20010040 Pbp_for
GTGCGTCATATGGAAGCAGAATCATCACATAAAACCGA 127 Pbp_rev
GCGTCTCGAGGGTGATTTTTTTGTTAATCACCCGTTG 128 Pap1_for
GTGCGTCATATGTTATCAGGACATTCGAGGTCA 129 Pap1_rev
GCGTCTCGAGAACAAACTTTTTATTAATAATCTCATTAAATTG 130 4883 Pbp_for
GTGCGTCATATGGAGACGGCAGGGGT 131 Pbp_rev GCGTCTCGAGAGTGTCACGCTTATTTGT
132 2724 Pbp_for GTGCGTCATATGAAAGATGATACTGCACAACT 133 Pbp_rev
GCGTCTCGAGTTCACCTAGCTTGGTGTTAG 134 Pap1_for
GTGCGTCATATGTACAGTAGATTGAAGAGAGAGTTAG 135 Pap1_rev
GCGTCTCGAGCTGATAAATCTTATAATTTTTAATCATG 136 2720 Ppb_for
GTGCGTCATATGGAGTACTGGTTCAATATTGAATGTTAAG 137 Pbp_rev
GCGTCTCGAGAGTGTCACGCTTATTTGTGACTG 138 Pap1_for
GTGCGTGCTAGCCAAAGCATATTTGGAGAGGAAAAGAG 139 Pap1_rev
ACTCGCTAGCGGCCGCAAGATCTTTTCGGTTTTCAAAAGCTAC 140 2728 Ppb_for
GTGCGTGCTAGCGAGACGGCAGGGGT 141 Pbp_rev GCGTCTCGAGAGTGTCACGGTTATTTG
142 Pap1_for GTGCGTGCTAGCCAAAGCATATTTGGAGAG 143 Pap1_rev
GCGTCTCGAGAAGATCTTTACGGTTTTCA 144 Dsm2071 Pbp_for
GTGCGTCATATGTTATCAAAAGATGATAAGGCGGAG 145 Pbp_rev
GCGTCTCGAGTTCACCTAATTTGGTGTTAGGAATTTC 146 6180 Pbp_for
GTGCGTCATATGACAGCTTCTTTAAATCAAAACGTAAAATCTGAG 147 Pbp_rev
GCGTCTCGAGTTGAGTGTCACGGTTATTTGTGAC 148 Pap1_for
GTGCGTCATATGGCTCACGAATTGGTTGAGGT 149 Pap1_rev
GCGTCTCGAGAAGATCTTTACGGTTTTCAAAAGTGAC 150 4538 Pap1_for
GTGCGTGCTAGCGCTCACGAATTGGTTGAGGTAC 151 Pap1_rev
GCGTCTCGAGAAGATCTTTTCGGTTTTCAAAAGTGAC 152 20010012 Pbp_for
GTGCGTCATATGGAGACTTTGCAGGACAGAAC 153 Pbp_rev
GCGTCTCGAGTTCAGGACGTTTGTTGTTTTCTAC 154 4959 Ppb_for
GTGCGTCATATGGAGACGGCAGGGGT 155 Ppb_rev GCGTCTCGAGAGTGTCACGCTTATTTGT
156 Pap1_for GTGCGTCATATGGCTGAAGAAAAATCTACTG 157 Pap1_rev
GCGTCTCGAGAAGATCTTTTCGGTTTTC 158 20010070 Pbp_for
GTGCGTCATATGGAAGTAAATTATGTAAAATCAGGAGTTATTG 159 Pbp_rev
GCGTCTCGAGAGTGTCACGCTTATTTGTGACTG 160
TABLE-US-00007 TABLE II FCT3 FCT2 M18, M33, FCT4 FCT6 FCT5 FCT1
FCT9 ID % M1 M3 M5, 44, 77 49 53 stD33 M9 M11, 78, 89 M12, 22, 50
M28 M2 M4 M6 M23 M75 FCT2 M1 100 39 39 36 39 38 39 38 40 40 21 23
23 20 24 FCT3 M3 >99 64 67 68 67 60 55 61 72 20 24 25 25 27 M5,
44, 77 >99 61 69 64 55 50 65 62 25 23 23 25 23 M18, 49 >90 65
65 58 54 62 65 24 23 24 23 25 M33, 53 >97 63 55 52 64 63 26 23
26 23 30 stD33 100 56 55 62 81 26 22 24 24 22 FCT4 M9 100 57 53 56
22 24 27 23 23 M11, 78, 89 >99 51 52 23 20 24 21 26 M12, 22, 50
>97 58 26 21 26 23 25 M28 >99 23 24 23 23 22 FCT6 M2 100 26
21 24 40 FCT5 M4 100 24 25 24 FCT1 M6 >99 55 23 M23 100 24 FCT9
M75 100
TABLE-US-00008 TABLE III SEQUENCE LISTING AND STRAINS FOR PBP SEQ
ID Strain 1 M1-SF370 2 M2-10270 3 M3-315 4 M4-10750 5 M5-Manfredo 6
M6-10394 7 M75-20010012 8 M9-2720 9 M11-2727 10 M12-A735 11
M18-8232 12 M23-DSM2071 13 M28-6180 14 M49-591 15 M33-29487 16
M1-5005 17 M12-2096 18 M12-9429 19 M12-2728* 20 M12-20010296* 21
M12-20020069* 22 M2-20010064 23 M2-20010065 24 M2-20010194 25
M2-20030561 26 M22-20020641 27 M22-20023465 28 M22-20023621 29
M28-20010164 30 M28-20010218 31 M28-20030176 32 M28-20030574 33
M28-20030902 34 M28-4436 35 M3-2721 36 M3-3040 37 M3-SSI 38
M4-20010040 39 M4-20010092 40 M4-20030968 41 M44-3776 42 M44-5481
43 M5-4883 44 M50-4538 45 M53-ALAB49 46 M6-2724 47 M6-3650 48
M6-D471 49 M77-4959 50 M78-3789 51 M89-20010070 52 M89-20021915 53
M89-20023717 54 M89-20030266 55 M89-20030382 56 STD633-D633 57
M1-3348 *Partial
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110195409A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110195409A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References